GE Healthcare
Application Note 28-9061-96 AA
Process Chromatography
Sanitization of ÄKTAprocess with
sodium hydroxide
This application note describes sanitization of
ÄKTAprocess™ systems after infection with baker’s yeast
(Saccharomyces cerevisiae). The three systems tested
were constructed of 10 mm polypropylene (PP); 3/8” and
1” stainless steel (SS), respectively.
The systems were infected with a solution containing
approximately 1 × 106 viable organisms/ml of baker’s
yeast. The infected system was incubated for 16 to 20 h
at room temperature, and then rinsed with purified water.
Sanitization was performed by filling the system with
1 M NaOH at 75% of maximal system flow rate and then
allowing an additional 1 h contact time (without flow). The
efficiency of NaOH as sanitizing agent was evaluated by
sampling at predetermined points in the system.
Each of the three systems was examined for colony
forming units of baker’s yeast at 57 sampling points and
flowthrough was examined before and after infection.
All inspected sampling sites were visually clean after
sanitization and the results showed that 1 M NaOH
effectively sanitized ÄKTAprocess of baker’s yeast.
Introduction
ÄKTAprocess is an automated liquid chromatography
system built for process scale-up and large-scale
biopharmaceutical manufacturing. The system is available
in three flow rate ranges that extend up to 1800 l/h for large
volume manufacturing and can be constructed in either
electropolished stainless steel or polypropylene, depending
on process conditions and plant requirements.
Microorganisms, such as bacteria and yeasts are found
in many laboratory and production environments and
multiply rapidly after incubation. System sanitization is
important since regulatory authorities have high standards
for purity and low microbial presence in products used
imagination at work
in clinical applications. Therefore, the efficiency of the
sanitizing method must be evaluated and this can be
done by microbial challenge tests. Sodium hydroxide
is frequently used to sanitize both chromatography
equipment and media since it is documented as an effective
sanitizing agent, is inexpensive, and readily available.
Microbial challenge tests
The principle of microbial challenge tests is to introduce
a high concentration of microorganisms into the
equipment or media and then treat with a sanitizing
agent. After a specified length of time, samples are taken
at predetermined sites, and the numbers of surviving
organisms are counted.
In this study, baker’s yeast (Saccharomyces cerevisiae)
was selected as a challenging organism since yeast cells
are more robust than other frequently used cells, such as
Escherichia coli and Chinese Hamster ovary cells. After each
infection, the three systems were sanitized by 1 M NaOH,
rinsed, and microbial samples were taken at predetermined
sites.
Sanitization and other terms
Sanitization, defined as the use of chemical agents to
reduce microbial populations, is commonly used in
chromatography systems to maintain microbial presence
at a level that will minimize the risk of contaminating the
product being processed. Note that a very low microbial
level is generally acceptable to process operators
and regulatory bodies, who do not require systems
or equipment to be sterile or sterilizable. Sanitization,
sterilization and other related terms are explained in
Table 1.
Table 1. Explanation of relevant and related terms
Sanitization
The use of any chemical agent to reduce a microbial population.
Sterilization
The destruction or elimination of all forms of microbial life.
Disinfection
The destruction of potential pathogens.
Antimicrobial agent
An agent that minimizes or destroys microorganisms in vitro. The term antimicrobial is general and all-inclusive: antimicrobials
include sanitizers, sterilizers and disinfectants. However, sanitizers, sterilizers and disinfectants are not necessarily the same and
sanitization, sterilization and disinfection are not interchangeable terms.
Materials and Methods
Microbial sampling
Preparation of the test organism
Microbial samples were taken at 57 predetermined
sampling points (Fig 1 and Table 2); flowthrough samples
were also taken at infection; post-infection, and postsanitization (Table 3). The following methods were used to
estimate the numbers of challenging organisms:
For the microbial challenge test, baker’s yeast (25%
dry wt.) was diluted with sterile 50 mM NaCl to an
approximate concentration of 1 × 106 viable organisms/ml.
The concentration was determined by viable count (see
sampling method 4 in Microbial sampling).
Preparation and infection of the system
The system was prepared for sanitization by connecting
manifolds at the system inlets, outlets and column in/out.
The inlet manifolds were then connected by tubing to
the CIP inlet valves and tanks filled with sanitization and
rinsing solutions were connected to the CIP inlet valves (Fig
1). All tubing had metal fittings since some plastics have
low chemical resistance to 1 M NaOH. Prior to sanitization,
the filter was removed. Note that if biomass film is found
at the bottom of the filter housing, we recommend that
the surface is sprayed with 70% ethanol and wiped clean.
Finally, the pH electrode was replaced with a plug to avoid
damage at extreme pH.
The detachable parts of the system were disassembled and
sprayed with 70% ethanol before reassembling. The whole
system was then filled with 1 M NaOH and allowed to stand
still for 16 to 18 h and then rinsed with purified water until
pH was neutral. The challenge organism suspension was
pumped into the system at a flow rate between 33% and
50% of maximal flow rate. The air trap was filled to ordinary
operating level and the filter housing was in line (but
without filter). The systems were incubated without flow for
16 to 20 h at room temperature.
Sampling method 1 – direct filtration
Sample solutions (minimum 10 ml) were collected in sterile
tubes and then filtered through a 0.45 µm cellulose nitrate
membrane filter. The filter was washed with 100 ml sterile
0.9 mg/ml NaCl solution and then incubated on malt extract
agar plates at 30°C to 35°C for 5 d before the number of
CFU (Colony Forming Units) was counted and the number of
viable microorganisms in the sample was calculated.
Sampling method 2 – swab
Surface samples were taken with alginate swabs. The swab
was inserted into the tube containing the isotonic swab
rinse solution. After dissolution, the whole solution was put
into molten malt extract agar and allowed to solidify in Petri
dishes. The plates were incubated at 30°C to 35°C for 5 d
before the number of CFU was counted and the number of
viable microorganisms in the sample was calculated.
Sanitization procedure
Sampling method 3 – peptone water filtration
Detachable parts were aseptically removed and put in
50 ml sterile peptone water in a 250 ml Ehrlenmeyer flask.
The flask was vigorously shaken for at least 20 min and
then the peptone water was filtered through a 0.45 µm
cellulose nitrate membrane filter. The filter was washed with
100 ml sterile 0.9 mg/ml NaCl solution and then incubated
on malt extract agar plates at 30°C to 35°C for 5 d before
the number of CFU was counted and the number of viable
microorganisms in the sample was calculated.
After incubation, the system was rinsed with purified water
and filled with 1 M NaOH at 50 to 75% of maximal system
flow rate. The air trap and the filter housing were over-filled.
After the filling with NaOH, the system stood without flow
for 1 h. The procedure was finished by rinsing with sterile
water to achieve neutral pH before sampling.
Sampling method 4 – viable count
Samples of the yeast solution, infection sample, and
flowthrough were diluted and incubated on malt extract
agar plates at 30°C to 35°C for 1 to 2 d before the number of
CFU was counted and the number of viable microorganisms
in the sample was calculated.
2
Application Note 28-9061-96 AA
Flexible tubing (1–3)
Manifold
F
System
pump A
Air outlet valve,
manual
Air outlet valve,
controlled
Air trap
Filter
P
Air
Air sensor
Press.
C sensor
Cond.
cell
1
3
2
4
CIP/AxiChrom™ Column packing valves
Press.
sensor
System pump B
(gradient pump)
Press.
sensor
UV
cell
P
UV
ld
Manifo
Air
Flow
sensor meter
F
Air P
ld
Manifo
Manifold
Manifold
Flow
meter
C
pH
Cond. pH
cell cell
Column valves
(Column in and out connected by manifolds)
Manifold
P
Press. sensor
Sample pump
Fig 1. The ÄKTAprocess flow scheme.
Results
The results of the microbiological sampling that showed
that treatment with 1 M NaOH for 1 h efficiently sanitized all
the 57 sampling points in each system.
All inspected sampling sites were visually clean after
sanitization except for biomass in the bottom of the filter
housing (Table 2). Note that if biomass film is found at
the bottom of the filter housing, we recommend that the
surface is sprayed with 70% ethanol and wiped clean.
The results in Table 3 show that all three systems had
infection and post-infection solutions with approximately
1 × 106 viable organisms/ml of baker’s yeast. After
sanitization treatment the concentration of challenging
organisms in the flowthrough solutions was zero.
Table 2. Number of CFU of baker´s yeast remaining after sanitization
No. of
sampling points
Inlet valve
System pump A
Cond. cell
Press. sensor
Air trap
Filter housing
Air sensor
Column valve
pH-cell
UV flow cell
Outlet valve
Sampling method
6
7
4
5
16
4
1
2
4
2
6
10 mm PP
(CFU/ml or
CFU/sampled unit)
2, 3
2, 3
2, 3
2, 3
2, 3
2
2
2
2, 3
2
2, 3
0
0
0
0
0
01
0
0
0
0
0
3/8” SS
(CFU/ml or
CFU/sampled unit)
1” SS
(CFU/ml or
CFU/sampled unit)
0
0
0
0
0
01
0
0
0
0
0
0
0
0
0
0
01
0
0
0
0
0
∑ 57
1
visual biomass
Table 3. Number of CFU of baker’s yeast in flowthrough at different phases of sanitization
Sanitization phase
Infection/start concentration
Post-infection, flowthrough sample
Post-sanitization flowthrough sample
Sampling method
4
4
1
10 mm PP
S. cerevisiae
(CFU/ml)
3.4 x 106
4.0 x 106
0
3/8” SS
S. cerevisiae
(CFU/ml)
3.9 x 106
3.2 x 106
0
1” SS
S. cerevisiae
(CFU/ml)
1.9 x 106
3.1 x 106
0
Application Note 28-9061-96 AA
3
Conclusions
The results show that the three sizes of ÄKTAprocess
systems examined can be efficiently sanitized with
1 M NaOH and the recommended procedure after
contamination with high concentrations of baker’s yeast.
However, the sanitization method cannot alone guarantee
good hygienic status of a chromatographic process. It
should always be applied in conjunction with other welldesigned and carefully controlled hygienic routines, as
well as rigorous control of buffers, water, and other input
material.
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ÄKTAprocess and AxiChrom are trademarks of GE Healthcare
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of General Electric Company.
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contractual approval, if required, to make changes in specifications
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© 2006 General Electric Company – All rights reserved.
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imagination at work
28-9061-96 AA
10/2006